Introduction: Extracellular vesicles (EVs) are spherical structures, naturally secreted by Gram-negative and Gram-positive bacteria. EVs play a critical role in the modulation of immune responses, bioactive cargo delivery, and cell-cell communication. The conventional method of EVs preparation involves the use of detergent (ultracentrifugation method). For the first time, we used a polyethylene glycol (PEG)-based method in our study to isolate EVs from prokaryotic cells, namely Faecalibacterium prausnitzii A2-165. We then compared various features of this method with those of the ultracentrifugation method. Methods: Extraction of EVs was performed via sequential deoxycholate ultracentrifugation and PEG-based methods. The physicochemical properties of the extracted EVs were compared via scanning electron microscopy (SEM), SDS-PAGE, and dynamic light scattering (DLS). Results: The protein content of the extracted EVs was 1.6 and 0.5 mg/mL, based on the ultracentrifugation and PEG-based methods, respectively. According to the SDS-PAGE analysis, vesicle-associated proteins were located at 20-150 kDa. The SEM analysis showed that the extracted EVs had a diameter of 50-200 nm in both methods. The results of DLS analysis showed 4 populations of approximately 50-8000 nm in the ultracentrifugation method and approximately 100-2000 nm in the PEG-based method. The EVs extracted by the ultracentrifugation method showed higher negative charge densities in contrast to EVs extracted by the PEG-based method. Conclusion: Our result showed that PEG-based extraction is a fast, simple, and cost-effective method and EVs purity was within the acceptable range. Further studies are needed to confirm the safety and the efficacy of EVs in clinical practices, especially as vaccine delivery vehicles.